Non coding RNA

Question 1

miRNA brief function

Answer 1

microRNA, regulate gene expression via translational regulation of mRNAs/degradation of mRNA. Originate from genome

Question 2

siRNA brief function

Answer 2

small interfering RNA, regulate gene expression via cleavage of mRNA, transcriptional silencing, mobile element silencing. Formed from recognizing dsRNA’s (generally exogenous).

Question 3

Long non-coding RNAs

Answer 3

> 200 nucleotides, can be involved in silencing, regulation, particularly regulation of structural organization (only thing it does that small does not)

Question 4

Describe the biogenesis and function of miRNA

Answer 4

Biogenesis:
- Transcribed from genome into long precursors
- Processed into smaller hairpin
- Hairpin moves into cytoplasm
- Further changed by dicer and placed into loading complex with argonaute, guide strand stays (RISK)
Function:
- miRNA will bind mRNA (often 3’ UTR. 1 miRNA can target multiple mRNA) and regulate translation of the target mRNA.

Question 5

siRNA biogenesis

Answer 5

Biogenesis:
Smalled double-stranded RNA molecules (often viral) is cut into 22 small siRNA molecules by DICER. dssiRNA then joins with Ago to form RISC, and RNA becomes sssiRNA, so it can target mRNA sequences.

Question 6

Describe the role of Argonautes and their function

Answer 6

Argonautes〠 bind to miRNA or siRNA through loading complex (makes risc), have slicer, nuclease, and/or dicer activity to cleave nucleotide sequence

Question 7

Describe long non-coding RNAs and some known activities and cellular functions.

Answer 7

Majority of genome ~90% non coding. Forms complex network long RNAs with no protein capacity (lncRNA). Have been found to form complex three dimensional structures through base pairing.

Are involved in modifying chromatin, act as scaffolding, sequester proteins, regulate mRNA transcription, translation…

Question 8

Describe the efforts associated with using regulatory RNAs for treatment.

Answer 8

RNA therapeutics (antisense oligonucleotides)  have ability to tailor RNA sequence to target any mRNA transcript, and therefore any protein. Greater specificity can be achieved over pharmacologics relying on protein substrates. 
Potential uses include vaccines, tumor antigen, and protein replacement.

Question 9

Describe issues associated with using regulatory RNAs for treatment.

Answer 9

Issues include nucleic acids stability, delivery to target tissue, cell entry, and toxicity.

Question 10

Describe antisense oligonucleotide therapeutics

Answer 10

Complementary oligonucleotide sequences that can base pair target RNA and lead to degradation, sequestration, or inhibition of activity

Question 11

RNAse H-dependent antisense nucleotides

Answer 11

• DNA sequences that bind and lead to cleavage of target RNA (RNAse H recognize RNA DNA base pairing and degrade) and inhibition of translation by steric hindrance of ribosomes
• Challenges: stability, reduction of losses due to excretion etc, delivery to specific target tissue, entry into cell and toxicity

Question 12

Exon-skipping ASOs

Answer 12

• Specifically target splice sites, binding and forcing choice of an alternate splice site. Can use to circumvent nonsense mutation
• Alternate splice site can restore stability or function to mutated gene product (ie. “cystic fibrosis”(probably actually muscular dystrophy, dystrophin)

Question 13

Describe an example of a disease that might be treatable with RNAs, how it would be treated, and the limitations. (3)

Answer 13

• Cystic fibrosis (maybe muscular dystrophy); People with cystic fibrosis have antisense mutation that truncates RNA. By binding oligonucleotides to the splice site (Exon skipping ASO) it forces alternative splicing that removes the exon. Makes protein functionable.
• Also used for: Optic neuropathy, familial TTR amyloidosis